1. Field of the Invention
This invention relates to host cells expressing one or more prohormone convertase enzymes and the production of a biologically active polypeptide from these cells. The invention further relates to polypeptide precursor variants having endoprotease cleavage sites which are processed by the host cell.
2. Description of Related Art
Most, if not all proteinaceous hormones are synthesized as relatively large precursor molecules, prohormones, that are biologically inactive (reviewed in Docherty and Steiner 1982; Loh et al. 1984; Mains et al. 1980). Maturation of the prohormone to its active form often requires endoproteolytic cleavage at paired or multiple basic amino acid residues to liberate the active component from the inactive portion of the precursor molecule. Until recently, almost nothing was known concerning the identity of the proteins responsible for this important stage in processing: the prohormone convertase (PC) enzymes.
Not every kind of cell has the capacity to correctly convert a prohormone to its active mature form through these specific cleavages. For some classes of prohormone this processing is apparently limited to those cells that contain both constitutive and regulated pathways of protein secretion (Gumbiner and Kelly 1982). Cells having both constitutive and regulated pathways of protein secretion are located almost exclusively in the specialized hormone-producing tissues of the endocrine and neuroendocrine systems.
An example of a family of hormones that is processed during regulated secretion is the insulin family of hormones. This family includes insulin, the insulin-like growth factors IGF-I and IGF-II, and relaxin. In their native environment, all members of this family are synthesized as precursor molecules that require processing to yield active hormone.
When heterologously expressed in cells having only a constitutive pathway of protein secretion, most hormone precursors, such as human preproinsulin, are secreted in an unprocessed prohormone form (Gumbiner and Kelly 1982). Experimental manipulation of mouse AtT-20 cells that disrupted the regulated secretory pathway of those cells has been observed to redirect the polyhormone precursor proopiomelanocortin (POMC) into the constitutive secretory pathway. In that case, POMC was no longer subjected to processing and was found to be secreted from the cell as the intact precursor (Moore et al. 1983). These observations suggest that there is a class of processing enzymes that function only in the regulated pathway of protein secretion; this pathway is apparently limited to certain highly-specialized cell types.
The POMC protein is a prohormone that is subject to differential processing. Expression of mature POMC derivatives is highly tissue-specific; alternate processed forms of the same prohormone precursor are produced in different regions of the brain (Douglass et al. 1984). The enzyme(s) and control mechanisms involved in the generation of this diversity are unknown. The possibility exists that there are tissue-specific enzymes that recognize unique amino acid sites on the prohormone substrate, or alternatively, that only one enzyme is responsible for the endoproteolytic cleavage and is itself under tight metabolic control, with each tissue providing a characteristic intracellular environment that is associated with cleavage at a specific subset of residue pairs.
Until recently, the only known eukaryotic prohormone processing enzyme was the KEX2 gene product of the yeast Saccharomyces cerevisiae (Jullius et al. 1983 and 1984; Fuller et al. 1989a.) The kex2 protein is a serine protease related to the subtilisin family of enzymes and has a preference for specific pairs of basic amino acids on its native hormone precursor substrates (pro-a-factor mating type pheromone and the pro-killer toxin). Kex2 shows maximum enzymatic activity at neutral pH with a strict requirement for the presence of calcium (Julius et al. 1984; Fuller et al. 1989a). It is membrane-bound and the mature, active form of the enzyme is localized in the post-Golgi compartment of the yeast cell (Fuller et al. 1989b; Redding et al. 1991). It can effectively serve as a substitute convertase for bona fide mammalian PC enzymes when heterologously expressed in otherwise processing-deficient cells by its demonstrated ability to correctly process certain mammalian prohormones: Nerve growth factor, bNGF, in BSC-40 cells (Bresnahan et al. 1990); protein C in baby hamster kidney BHK cells (Foster et al. 1991); POMC both in BSC-40 cells (Thomas et al. 1988) and in COS-1 cells (Zollinger et al. 1990)]. Kex2 was shown to have a highly similar if not identical substrate specificity to the authentic human proalbumin convertase in vitro (Bathurst et al. 1986; Brennan et al. 1990). When heterologously expressed in mammalian cells kex2 will home to the post-Golgi compartment, where it is apparently fully active (Germain, et al. 1990). These observations have led to speculation that this yeast protein must be both functionally and structurally similar to an authentic mammalian convertase. A search began for the elusive mammalian counterparts of kex2 based upon structural homologies.
KEX2 and Fur Hydrophobic Anchor
The Kex2 endoprotease has two hydrophobic regions located at the N-terminal side and C-terminal side. The C-terminal hydrophobic transmembrane anchor is responsible for the anchoring of the Kex2 to a Golgi body of a yeast cell. Deletion of this C-terminal hydrophobic anchor renders the Kex2 endoprotease soluble while still maintaining substrate specificity (EPO PUB No.0327377).
An inspection of genetic data bases identified a potential mammalian homologue of kex2 that shared many features of the active site domain of the kex2 protein: the fur gene product of human liver, furin (Fuller et al. 1989b). Furin was subsequently cloned and successfully expressed in processing-deficient cells: cotransfection of furin with pro-von Willebrand factor in COS-1 cells (van den Ven et al. 1990; Wise et al. 1990) and with pro-bNGF in African green monkey kidney epithelial BSC-40 cells (Bresnahan et al. 1990) resulted in correct processing of the precursor substrates.
However, when furin and a prohormone, prorenin, have been coexpressed in mammalian cells, no processing has been observed (Hatsuzawa, et al The Journal of Biological Chemistry Vol 265 [1990]).
Furin also shared with kex2 a requirement for calcium ions, displayed maximum activity at a neutral pH, and, like kex2, was shown to be a membrane-bound protein in the post-Golgi compartment (Bresnahan et al. 1990). However, because furin does not seem to be capable of efficiently processing certain hormone precursors such as prorenin, and its mRNA message is apparently expressed in most if not all mammalian cells (Hatsuzawa et al. 1990), the furin protease may play a role in an essential xe2x80x9chousekeepingxe2x80x9d function in that it could be responsible for many of the basic amino acid site cleavages occurring in the constitutive secretory pathway of the cell. These functions could be general, or more confined to specific cell-types with the constitutive pathway-dependent processing of growth factors like bNGF.
Because furin does not appear to be directly involved in the endoproteolytic processing of prohormones in endocrine-like tissues, there must be other mammalian proteins both functionally and structurally similar to furin and yeast kex2 that serve as the authentic prohormone convertases: PC proteins that share distinctive homologies in their active sites. This search for structural homology has recently lead to the discovery of more PC proteins.
By using the technique of xe2x80x9cMixed Oligonucleotides Primed Amplification of cDNAxe2x80x9d (MOPAC, Innis 1990), Smeekens and Steiner (1990) were able to use the conservation of amino acid sequence surrounding the active sites of both bacterial subtilisin and the yeast kex2 protease to amplify a putative prohormone convertase cDNA from human insulinoma. This PC cDNA, termed mPC2, showed an exceptional degree of homology to the yeast kex2 protease. In similar experiments Seidah and colleagues identified another member of the subtilisin family of proteases, termed, mPC1 (Seidah, et al 1991).
The distribution of PC1 and PC2 has so far been observed to be confined to neuroendocrine-derived tissues (Seidah et al. 1990; Seidah et al. 1991; Smeekens et al. 1991), suggesting that these proteins may be candidates for the authentic convertases resident in the regulated secretory pathways of these tissues. The substrate specificities of PC1 and PC2 for defined pairs of dibasic amino acids at the prohormone cleavage sites do not appear to be identical (Benjannet et al. 1991; Thomas et al. 1991), nor do they share the same pattern of tissue distribution in the brain (Seidah et al. 1991). This implies that different classes of prohormones may require unique PC enzymes, and that both PC1 and PC2 may be members of a family of specific processing enzymes employed by the endocrine system to generate the diversity of hormones required throughout the entire organism.
Precursor processing
The biosynthetic process begins with the synthesis of the precursor (prepropeptide) on the rough endoplasmic reticulum (RER). The signal peptide (pre-portion) is clipped off as the proprotein is transported into the cisternae of the RER where protein folding, di-sulfide bond formation and asparagine-linked glycosylation occur (FIG. 1). The precursor is then translocated to the Golgi apparatus where more complex glycosylation and phosphorylation occur. Within the Golgi of some cells, proteins are sorted by an unknown mechanism into two groups: those that will be constitutively secreted and those that undergo regulated secretion. The constitutively secreted proteins will enter vesicles and be transported to their target continually without the need for any specific stimulus. Proteins undergoing regulated secretion are transported to secretory vesicles and will be released only when an adequate stimulus is provided. Within the secretory vesicles the excision of bioactive peptides from the larger inactive protein precursors occurs. Two steps involved in this process are endoproteolytic cleaveage usually at the carboxyl side of paired basic amino acids (e.g. lys-arg, arg-arg) and exoproteolytic cleavage of flanking basic amino acids by carboxyl- and/or aminopeptidases (reviewed in Mains et al. 1990.).
Many proteins, including NGF (Berger and Shooter 1977), are first synthesized as larger precursor proteins. The function of the precursor is still not well understood. One possible role of the precursor is to aid in protein folding (Steiner 1982, Selby et al. 1987, Wise et al. 1988). The precursor may also direct the protein to the proper location or pathway within the cell as is suggested by Sevarino and colleagues (1989), though this is not the case with the connecting peptide of insulin (Powell et al. 1988, Gross et al. 1990). Additionally, the precursor has been shown to have roles in gamma carboxylation of glutamic acid residues (Pan and Price 1985, Furie and Furie 1988), and regulation of the coordinate synthesis of multiple mature peptides from a single precursor polypeptide eg., POMC. (For review, see Douglass et al. 1984).
Relaxin
In the present invention, prorelaxin is used as a typical hormone precursor. The relaxin is first synthesized as a preprohormone precursor which undergoes specific processing to form the mature two-chain, dilsulfide-linked active relaxin. A major part of this processing requires endoproteolytic cleavage at specific pairs of basic amino acid residues. This specific processing does not occur when the precursor is heterologously expressed in cells containing only the constitutive pathway of protein secretion. Mature human relaxin in an ovarian hormonal peptide of approximately 6000 daltons in molecular weight known to be responsible for remodeling the reproductive tract before parturition, thus facilitating the birth process. Hisaw, F. L., Proc. Soc. Exp. Biol. Med., 23:61-663 (1926); Schwabe, C. et al., Biochem. Biophys. Res. Comm., 75: 503-570 (1977); James, R. et al., Nature, 267: 544-546 (1977). This protein appears to modulate the restructuring of connective tissues in target organs to obtain the required changes in organ structure during pregnancy and parturition. Some of the important roles for relaxin as a pregnancy hormone include inhibition of premature labor and cervical ripening at parturition. While predominantly a hormone of pregnancy, relaxin has also been detected in the non-pregnant female as well as in the male. Bryant-Greenwood, G. D., Endocrine Reviews, 3: 62-90 (1982) and Weiss, G., Ann. Rev. Physiol., 46: 43-52 (1984).
Relaxin consists of two polypeptide chains, referred to as A and B, joined by dusulfide bonds with an intra-chain disulfide loop in the A-chain in a manner analogous to that of insulin. Two human genes (H1 and H2) for human relaxin have been identified, and only H2 is expressed in the ovary. Porcine relaxin, the sequence of which has also been determined, has been used in human clinical trials for ripening of the cervix and induction of labor. MacLennan et al., Obstetrics and Gynecology, 68: 598 (1986).
European Pat. Publ. No. 86,649 published Aug. 24, 1983 discloses how to prepare procine preprorelaxin, porcine prorelaxin, and porcine relaxin. Australian Pat. No. 561,670 issued Aug. 26, 1987, European Pat. Publ. No. 68,375 published Jan. 5, 1983, and Haley et al., DNA, 1: 155-162 (1982) disclose how to prepare porcine relaxin. European Pat. Publ. Nos. 101,309 published Feb. 22, 1984 and 112,149 published Jun. 27, 1984 respectively disclose the molecular cloning and characterization of a gene sequence coding for human relaxin and human H2-relaxin and analogs thereof. U.S. Pat. No. 4,267,101 issued May 12, 1981 discloses a process for obtaining human relaxin from fetal membranes.
Nerve Growth Factor
Nerve growth factor (NGF), required for sympathetic and sensory neuron survival (Levi-Montalcini and Booker 1960, Gorin and Johnson 1979), is the most well characterized neurotrophic factor in part due to the exceptionally high levels synthesized in the male mouse submaxillary gland. It was from this source that NGF was purified (Cohen 1960) and its complete amino acid sequence determined (Angeletti 1973). Using this information, Scott and colleagues (1983) identified and cloned the mouse NGF gene by screening a mouse submaxillary gland cDNA library using a probe constructed for a hexapeptide based on the least degenerate codons contained within the NGF sequence. Molecular cloning, using the polymerase chain reaction (PCR) (Saiki et al. 1985, Mullis et al. 1986), has recently revealed that NGF is a member of a family of neurotrophic factors that also includes BDNF (Leibrock et al. 1989) and NT-3 (Hohn et al. 1990, Rosenthal et al. 1990, Maisonpierre et al. 1990a, Ernfors et al. 1990, Jones and Reichardt 1990).
NGF, BDNF and NT-3 are all translated as preproproteins that require endoproteolytic cleavage in order to be active. There is an extensive amount of homology ( greater than 50%) between the mature portions of NGF, BDNF, and NT3. However, the homologies of the precursor portions are much lower (xcx9c20%). The efficiency of processing of the three neurotrophic factors, from their inactive pro-form to the active factor, vary substantially with different cell types.
Insulin
Insulin is a polypeptide hormone which is produced in the beta cells of the islets of Langerhans situated in the pancreas of all vertebrates. Insulin is secreted directly into the bloodstream where it regulates carbohydrate metabolism, influences the synthesis of protein and of RNA, and the formation and storage of neutral lipids, The Merck Index, 10th edition, 1983 Insulin promotes anabolic processes and inhibits catabolic ones in muscle, liver and adipose tissue. The structure of human insulin was disclosed in Nature 187,483 (1960). Prior to the discovery of recombinant DNA technology, the major source of insulin for human consumption was the pancreases of slaughtered animals. Human insulin was among the first commercial health care products produced by recombinant technology. A review of the research, development, and recombinant production of human insulin is in Science 219, 632-637 (1983).
Glucose regulation of insulin responsiveness
Circulating insulin levels are regulated by several small molecules including glucose, amino acids, fatty acids and certain pharmacological agents. (Selden, et al., Nature vol. 321 pg 525-528 [1986]). When ambient glucose is detected by the a and b cells of pancreatic islets, they consequently secrete either glucagon, which stimulates glucose release from liver, or insulin, which induces glucose storage in liver and stimulates glucose uptake by muscle and adipocytes. (Thorens et al, Diabetic Care, vol. 13, no. 3, pgs. 209-218 [1990]).
Glucose stimulates de novo insulin biosynthesis by increasing transcription, mRNA stability, translation, and protein processing. Glucose also rapidly stimulates the release of pre-stored insulin. While glucose and non-glucose secretagogues may ultimately work through the same pathway, the biochemical events leading from changes in the levels in a particular fuel to insulin secretion are initially diverse. In the case of glucose, transport into the b-cell and metabolism are absolute requirements for insulin secretion. (WO 92/21756, published Dec. 10, 1992).
The cellular uptake of glucose is accomplished by membrane-associated carrier proteins that bind and transfer it across the lipid bilayer. Two classes of glucose carriers have been identified in mammalian cells, the Na+-glucose cotransporter and the facilitative glucose transporter (GLUT). Five members of the GLUT family have been identified: GLUT 1, expressed in erythrocytes; GLUT 2, expressed in liver; GLUT 3, expressed in brain; GLUT 4, expressed in muscle and fat cells; and GLUT 5, expressed in small intestine. (Bell et al., Diabetes Care, vol. 13, no. 3, pg. 198-208 [1990]). GLUT 2 is unique among the five member family of glucose transporters in that it has a higher Km and Vmax for glucose. (WO 92/21756, published Dec. 10, 1992).
GLUT 2 and the phosphorylating enzyme, glucokinase have been implicated in the control of glucose metabolism in islet b cells. Glucokinase is the high Km and high Vmax counterpart of GLUT 2 among the family of hexokinases. GLUT 2 and glucokinase have been hypothesized to be the glucose sensing apparatus that modulates insulin secretion in response to changes in circulating glucose concentrations by regulating glycolytic flux. (WO 92/21756, published Dec. 10, 1992).
Hexokinase performs the same function as glucokinase (glucose phosphorylation) but does so at much lower glucose concentrations (hexokinase has a Km for glucose of approximately 0.05 mM versus a 8 mM for glucokinase).
Cuif et al., (Mol Cell Biol 12 (11) p4852-61 [1992]), report identification of a glucose-response element of an enzyme, L-type pyruvate, in transgenic mice. Cuif et al., Supra indicate that the proximal region between xe2x88x92183 and +11 bp confers tissue-specificity and contains all the information necessary for the dietary and hormonal control of the L-PK gene expression in vivo, and further identify distal sequences that modulate transcriptional activity in a tissue specific manner.
In addition to its role in vivo, insulin is also useful in recombinant cell culture. Insulin is an example of a polypeptide factor important for mammalian cell culture proliferation and anabolism. Some cell cultures produce endogenous insulin and some do not; cell cultures which rely on added insulin are problematic because insulin is unstable in some cultures. European publication number, 0307247A2, published Mar. 3, 1989, describes the introduction of nucleic acid encoding insulin into a mammalian host cell to eliminate the need for adding exogenous insulin.
Insulin is synthesized as a larger precursor protein, proinsulin. Proinsulin is a single polypeptide chain containing a sequence of about thirty residues that is absent from mature insulin. Proinsulin, like prorelaxin, has a B-C-A chain structure. The C or connecting peptide joins the carboxyl end of the B chain and the amino terminus of the A chain of the future insulin molecule Biochemistry 3rd edition, pg. 995 (1988). The mature insulin is generated by cleavage of the C peptide at dibasic residues Arg(31)-Arg(32) and Lys(64)-Arg(65). Two distinct processing enzymes have been defined which are specific for their respective dibasic cleavage sites in proinsulin; type I is substrate specific for the BC junction, while type II is specific for the CA junction (Weiss, Biochemistry 29, 1990).
Naturally occurring mutations in the human insulin gene have been reported by Steiner et al. (Diabetes Care vol. 13, no. 6 pg. 600-609 [1990]). Members of a family with hyperproinsulinemia have a substitution of insulin B chain residue 10, a histidine, with aspartic acid resulting in a proinsulin that is reported to exhibit altered subcellular sorting behavior. In patients having hyperproinsulinemia, a significant proportion of the newly synthesized Asp-10 proinsulin is secreted from the islets in an unprocessed form via an unregulated or constitutive protein secretory pathway (Steiner et al. PNAS USA, vol. 85, pg. 8943-8947 [1988]) and (Quinn et al. The J. Cell Bio, vol. 113, pg. 987-996 [1991]). Others have shown that insulin containing this B10 H greater than D mutation results in a more active form of insulin with increased binding to the insulin receptor (Schwartz, et al. PNAS USA vol. 84, pp. 6408-6411 [1987]; Brange et al. Nature vol 333 pg 679-682 [1988]; Shoelson et al. vol. Biochem. vol. 31 pg 1757-1767 [1992]). Brems et al. (Protein Engineering vol. 5 no. 6 pg 519-525 [1992]) report that the replacement of B-chain residue 10, a histidine, with aspartic acid increased the stability of the insulin. Wild-type human insulin exhibits subunit interactions and forms dimers; dimeric insulin binds to Zn2+ to form hexamers. The histidine residue at position B10 in insulin is involved in co-ordinating zinc ions and it has been reported by Quinn et al. Supra, that the human insulin mutant, B10 histidine to aspartic acid, allows formation of dimers of human proinsulin but not hexamers.
Insulin Like Growth Factor I and II (IGF-I and IGF-II)
Insulin-like growth factors, or IGFs, by definition are polypeptides with insulin-like structural and biologic properties which are not neutralized by the presence of excess anti-insulin antibodies. (Endocrinology and Metabolism 2nd ed. [1987]). The complete amino acid sequences of IGF-I and IGF-II have been determined (Rinderknecht, et al Journal of Biological Chemistry 253 pg. 2769 [1978]; Rinderknecht et al FEBS Letters 89, 283 [1978]). They are both single-chain polypeptides with three disulfide bridges and a sequence identity of 49 and 47 percent respectively, to human insulin A and B chains. The connecting peptide or C region is considerably shorter than the one of proinsulin and does not show any significant homology to it. The IGF-I peptide is not cleaved during the processing to the mature molecule. In addition, IGF-I contains a short, 8 amino-acid, carboxyterminal extension peptide, termed the D domain, for which no homologous region exists in insulin (Foyt, H. L., Insulin-Like Growth Factors: Molecular and Cellular Aspects [1991])
Both IGF-I and IGF-II are derived from precursors by proteolytic processing (Jansen et al Nature 306, 609 [1984]; Bell, et al Nature 310, 775 [1984]; Jansen et al FEBS letters 179, 243 [1985].
There exists a need for a method of producing polypeptides from their polypeptide precursors in cell culture. It is therefore an object of the present invention to provide host cells that express active prohormone convertases that cleave polypeptide precursors to polypeptides. It is also an object of the present invention to provide a method for the production of a desired polypeptide in cell culture in a manner that results in the proper processing and glycosylation of the desired polypeptide. A related objective of the present invention is the production of polypeptide hormones, such as insulin, relaxin, and IGF-I.
A further object of the present invention relates to providing polypeptide precursor mutants having prohormone convertase cleavage sites for processing by the host cell.
There also exists a need to eliminate problems associated with supplying necessary polypeptide factors (e.g. insulin or transferrin) for the maintenance and growth of recombinant host cells. This need is particularly evident in the use of mammalian cell culture in the production of commercial polypeptides such as pharmaceutical products. It is therefore an object of the present invention to provide mammalian cell cultures that express prohormone convertases that enable processing of polypeptide factor precursors to polypeptide factors needed for mammalian cell culture proliferation and anabolism. It is an object of the present invention to produce polypeptide factors using host cells expressing prohormone convertases.
These and other objects of the invention will be apparent to the ordinary artisan upon consideration of the specification as a whole.
The present invention describes a method for the production of a desired polypeptide in host cells expressing a prohormone convertase. In one embodiment the desired polypeptide is a prohormone processed to active hormone. In another embodiment, the desired polypeptide is any polypeptide factor needed for cell growth or anabolism. In some embodiments, the invention provides polypeptide factor mutants comprising as cleavage site recognizable by a host cell enzyme, such as a prohormone convertase. In a preferred embodiment, a prohormone convertase precursor is provided which is modified to have a host cell cleavage site allowing for processing of the prohormone convertase precursor to active enzyme in the host cell.
In one embodiment the host cell constitutively expresses a prohormone convertase. In another embodiment, the host cell is stably transformed with a prohormone convertase.
In one aspect of the present invention the production of a heterologous polypeptide factor, such as insulin or transferrin, in a polypeptide factor-depending host cell is accomplished by a) introducing into the polypeptide factor-dependent host cell nucleic acid encoding a heterologous polypeptide factor precursor comprising a cleavage site recognizable by a host cell enzyme and wherein said host cell is dependent on the cleavage product of said polypeptide factor precursor; and b) culturing said host cell under conditions wherein the polypeptide factor precursor is cleaved at said cleavage site by the host cell enzyme, thereby producing said polypeptide factor.
In another aspect of the present invention the production of a desired polypeptide in a host cell expressing a prohormone convertase is accomplished by a) introducing into the host cell nucleic acid encoding a desired polypeptide; and b) culturing said host cell under conditions wherein said desired polypeptide is expressed. Preferably the desired polypeptide is a polypeptide hormone and may be any polypeptide hormone. Preferably, it may be any polypeptide hormone comprised of two or more polypeptide chains. Among the preferred two-chain hormones are insulin, relaxin, and insulin-like growth factors I or II. Among the preferred prohormones are proinsulin, prorelaxin, and precursors of insulin-like growth factors I or II. The prohormone may be a prohormone mutant modified to contain one or more prohormone convertase cleavage sites. The prohormone or prohormone mutant may be processed by the prohormone convertase in an in vivo or in vitro manner.
The method of producing polypeptide hormones may be further accomplished by inserting into a prohormone, a prohormone convertase cleavage site that facilitates processing by a prohormone convertase. The preferred hormone is a mammalian polypeptide hormone comprised of two or more polypeptide chains, for example insulin, relaxin, insulin-like growth factor I or insulin like growth factor II.
In another aspect, the method may be practiced using cells transformed to contain a prohormone convertase fused to a hydrophobic transmembrane Golgi anchor, such as that from kex2 or furin.
The present invention discloses nucleic acid (a) encoding murine prohormone convertases 1 and 2, (b) mutants of prohormone convertase that contain inserted convertase cleavage sites, and (c) mutants of prohormone convertase that contain hydrophobic anchor domains, or heterologous pre and prepro sequences from other processed polypeptides. Also disclosed are vectors containing such nucleic acid and cells expressing such nucleic acid. Methods of effecting transformation and methods of providing transformed mammalian cells are disclosed.